1. Field of the Invention
The present invention relates to methods for assaying GABA-modulatory compounds for activity as antidepressants, cognitive enhancers, sedative hypnotics, or non-sedating anxiolytics. In particular, the method includes determining efficacy (generally in vitro efficacy) and EC50 values (as used herein incorporating IC51 values) for the compounds at several different cloned (i.e., expressed in cells as directed by heterologous cloned receptor-encoding nucleic acid expression vector molecules) GABAA subtype receptors (each subtype made up of a defined set of specific receptor subunit isotypes). The method optionally includes determining binding affinity of compounds for GABAA receptors. The method results in the development of an activity profile for each compound. As an additional step, animal models predictive of such effects may be used to measure the ability of compounds to effect cognitive enhancement, to act as antidepressants, to mediate sedative hypnotic effects, or to effect anxiolysis in vivo without eliciting certain undesirable side effects.
2. Description of Related Art
Modern drug discovery methodology allows the testing of large numbers of compounds (often assembled into collections termed libraries) for functional characteristics that confer pharmaceutical utility. This “screening” of such libraries, using specific tests (assays) for functional activity properties, allows the rapid identification of promising compounds for further development as pharmaceutical agents. There has been a longstanding quest in the pharmaceutical industry for new means of identifying such promising compounds. Such new means may involve new assays, or may use old assays to generate data that can be analyzed and applied in new ways to identify compounds with new and useful characteristics.
In the field of psychopharmacology, the use of cloned neuronal receptors as substrates has provided new, more specific assays with which compounds can be characterized. The use of such receptors has enabled the development of receptor binding profile criteria that are particularly beneficial in the identification of useful psychopharmacological agents. For example, such profiling can identify compounds that will be free of certain undesirable adverse effects (side effects).
The GABAA receptor superfamily represents one of the classes It of receptors through which the major inhibitory neurotransmitter, γ-aminobutyric acid, or GABA acts. In addition to being the site of neurotransmitter action a number of drugs including the anxiolytic and sedating benzodiazepines bind to this receptor. The GABAA receptor is a chloride channel that opens in response to GABA, allowing chloride to enter the cell. This effects a slowing of neuronal activity through hyperpolarization of the cell membrane potential. GABAA receptors are composed of several protein subunits and are generally pentameric in structure.
A number of cDNAs for GABAA receptor subunits have been cloned. While these subunits share a basic motif of 4 membrane-spanning helices, there is sufficient sequence diversity to classify them into several groups. To date at least 6α, 3β, 3γ, 1ε, 1δ, 2ρ, and 1π subunit species have been identified; some representing alternatively spliced forms. Native GABAA receptors are typically composed of α, β, and γ subunits, most often in the ratio of two alphas, two betas, and one gamma, although other combinations (some comprising other subunits such as ε, δ, ρ, or π) have been described. Even if restricted to only α, β, and γsubunits, however, an enormous diversity of GABAA subtype receptors are possible. Evidence such as message distribution, genome localization and biochemical studies suggests that the major naturally occurring receptor combinations are α1β2γ2, α2β3γ2, α3β3γ2, and α5β3γ2.
In the typical GABAA receptor, the binding sites for GABA (2 per receptor complex) are formed by amino acids from the α and β subunits. Amino acids from the α and γ subunits contribute to form 1 benzodiazepine site per receptor complex. In a classic allosteric mechanism, the binding of a drug to the benzodiazepine site increases the affinity of GABA binding to the receptor. Benzodiazepines and related drugs that enhance the ability of GABA to open GABAA receptor channels are known as agonists or partial agonists depending on the level of enhancement. Other classes of drugs such as β-carboline derivatives that occupy the same site and negatively modulate the action of GABA are called inverse agonists. A third class of compounds exists that occupies the same site as both the agonists and inverse agonists (blocking access of these agents to the site) and yet has little or no direct effect on GABA activity. These compounds are referred to as antagonists.
The characterization of activities of different subtype receptors has been an area of intense pharmacological discovery. Agonists that act at the benzodiazepine site have long been known to know to exhibit anxiolytic, sedative, and hypnotic effects in animal behavior models, while compounds that act as inverse agonists at this site elicit anxiogenic, cognition enhancing, and proconvulsant effects. While benzodiazepines have long been used as anxiolytics, these compounds exhibit a number of undesirable side effects. These include cognitive impairment, sedation, ataxia, potentiation of ethanol effects, and a tendency for tolerance and drug dependence. Likewise the development of benzodiazepine site ligands for other indications has been if thwarted by unfavorable side effect profiles for each indication. For example, compounds known to possess cognition enhancing properties have generally tended to be anxiogenic and proconvulsant, while compounds that produce anxiolytic effects tend to generate unwanted sedation, and do so more powerfully when taken in conjunction with the consumption of alcoholic beverages.